PROJECT SUMMARY Metastatic cancer is difficult to treat and often incurable. Characterizing pro-metastatic cellular pathways is therefore crucial for identifying new therapeutic targets and biomarkers for metastasis to improve clinical prognosis of cancer patients. Dysregulated cellular metabolism is a well-established hallmark of cancer, but a growing body of research highlights metabolic differences between primary and metastatic cancer cells. While highly proliferative primary tumor cells often exhibit the Warburg effect, marked by a preference for aerobic glycolysis to generate ATP, genes for mitochondrial biogenesis and oxidative phosphorylation are reported to be upregulated in metastatic cancer cells. Therefore, understanding the differential roles of mitochondrial metabolic pathways and enzymes in disseminated cancer cells is crucial for determining factors that give cancer cells a metastatic advantage. In order to colonize distant organs, cancer cells must first resist anoikis: an apoptotic cell death mechanism triggered by loss of proper contact with the extracellular matrix. Our lab has previously demonstrated that the mitochondrial enzyme glutamate dehydrogenase 1 (GDH1) contributes to anoikis resistance and metastasis by regulating the bioenergetic response through reactivation of AMPK in LKB1-deficient lung cancer. However, the role of other mitochondrial enzymes in anoikis resistance remains poorly understood. To identify other factors important for cancer cell anoikis resistance, we performed an unbiased RNAi screen targeting 120 mitochondrial enzymes in lung cancer cells and identified the ATP-specific Succinyl-CoA Synthetase beta subunit (SUCLA2) as a factor that may be important for cancer cell survival after ECM detachment. Stable knockdown of SUCLA2 sensitized multiple cancer cell lines to anoikis when cultured under non-adherent conditions in vitro. Bioinformatic analysis of publicly available data indicates that higher tumor SUCLA2 mRNA expression is associated with poor patient survival, and our immunohistochemistry staining suggests that SUCLA2 protein levels are higher in metastatic lung cancer compared to matched primary tumors. Additionally, our preliminary data suggests that SUCLA2 knockdown significantly increases oxygen consumption rates (OCR) and reactive oxygen species (ROS) levels in ECM detached cancer cells. In this proposal, we will employ multiple approaches to test our hypothesis that SUCLA2 promotes anoikis resistance and tumor metastasis by modulating cellular redox status following ECM detachment. The specific aims of the proposed research are 1) To determine whether SUCLA2 promotes anoikis resistance and metastasis of cancer cells in an enzyme-dependent manner and 2) To decipher the molecular mechanism by which SUCLA2 contributes to cellular metabolism to promote cancer cell anoikis resistance. In the long term, we seek to comprehensively characterize the metabolic pathways underlying SUCLA2's role in cancer cell anoikis resistance, which will be critical to providing a greater understanding of the metabolic signaling mechanisms underlying metastatic progression.